Combinatorial actions of IL-22 and IL-17 drive optimal immunity to oral candidiasis through SPRRs

Oropharyngeal candidiasis (OPC) is the most common human fungal infection, arising typically from T cell immune impairments. IL-17 and IL-22 contribute individually to OPC responses, but here we demonstrate that the combined actions of both cytokines are essential for resistance to OPC. Mice lacking IL-17RA and IL-22RA1 exhibited high fungal loads in esophagus- and intestinal tract, severe weight loss, and symptoms of colitis. Ultimately, mice succumbed to infection. Dual loss of IL-17RA and IL-22RA impaired expression of small proline rich proteins (SPRRs), a class of antimicrobial effectors not previously linked to fungal immunity. Sprr2a1 exhibited direct candidacidal activity in vitro, and Sprr1-3a-/- mice were susceptible to OPC. Thus, cooperative actions of Type 17 cytokines mediate oral mucosal anti-Candida defenses and reveal a role for SPRRs.


Introduction
The commensal pathobiont Candida albicans is the most frequent cause of human fungal infections, yet studies of fungal immunity lag considerably behind other microbes [1].Oropharyngeal candidiasis (OPC) ranges from a nuisance to a severe and painful condition, with potential to cause nutritional deficits or esophageal cancer [2][3][4][5].To date, there are no vaccines to C. albicans or indeed to any fungi, highlighting a need to better define the correlates of immunity required to restrain fungal pathogenesis [1,6].
We report that mice lacking the IL-17 and IL-22 receptors are exquisitely susceptible to OPC, more than either knockout alone.Infection is associated with up-regulation of small proline-rich (SPRR) proteins, a poorly defined class of antimicrobial effectors [20,21].We show that SPRRs exert direct candidacidal activity and contribute cooperatively to OPC immunity.
Esophageal fungal loads were higher in Il22ra1Il17ra -/-mice compared to Il22ra -/-mice, but were not statistically different from Il17ra -/-animals (Fig 1D).Il22ra1Il17ra -/-mice showed reduced colon lengths (Fig 1E ), indicative of intestinal tissue damage.However, fungal loads in the SI and colon were the same in all mouse strains (Fig 1D ), collectively suggesting that the oral cavity shows a particular sensitivity to combined actions of these cytokines.There was no fungal dissemination to visceral organs (kidney, liver, and spleen) (Fig 1F ), likely ruling out systemic candidiasis as cause of mortality.Thus, IL-17 and IL-22 act cooperatively to limit oral and to some extent esophageal candidiasis, but not intestinal colonization of this fungus.

The SPRR family is implicated in oral candidiasis
To understand how IL-22 and IL-17 act in OPC, we compared oral mRNA profiles after OPC in WT versus Il22ra1Il17ra -/-mice, revealing unique and overlapping expression patterns (Fig 2A).We focused on gene changes in Il22ra1Il17ra -/-mice immediately prior to severe morbidity (day 7), where 1,446 genes were expressed in Il22ra1Il17ra -/-compared to WT controls (Fig 2B ).A gene set not previously linked to antifungal immunity encoded antimicrobial small proline-rich proteins (SPRRs) (Fig 2C and 2D) [21,24,25].Sprr expression was negligible at baseline but induced in WT mice at early time points (day 2).By day 7 when C. albicans was cleared, SPRRs were no longer evident.In Il22ra1Il17ra -/-mice, SPRRs were impaired at day 2 though up-regulated at day 7, commensurate with high fungal burdens.IF staining confirmed oral expression of a representative SPRR, Sprr2a1, in WT mice at day 2 but not Il22ra1Il17ra -/- mice (Fig 2E).

SPRR2a mediates anti-Candida responses
To ascertain whether SPRRs have antifungal properties, C. albicans cells were cultured in vitro with recombinant SPRR2A1 and surviving fungi enumerated.There was a dose-dependent candidacidal effect of Sprr2A1 (Fig 3A and 3B).In vivo, mice lacking 3 Sprr2a genes (Sprr2a1, Sprr2a2, and Sprr2a3) were significantly but modestly susceptible to OPC, showing elevated fungal loads and increased percentages of mice infected (Fig 3C and 3D).However, OPC was not as severe in SPRR-deficient mice as in IL-17RA or IL-22RA knockouts, given the low fungal loads and full recovery from infection-induced weight loss (Fig 3C).Thus, SPRR family members are up-regulated in OPC and exhibit direct antifungal activity (Fig 3E ), though clearly additional antifungal pathways are also operative.

Discussion
These data show that IL-22R/IL-17R deficiency causes severe susceptibility to OPC, far more than loss of either cytokine receptor alone (Fig 3).Synergistic activities of IL-17 and IL-22 on target cells (typically nonhematopoietic) has been described previously, and particularly relevant here is cooperative up-regulation of antimicrobial effectors (β-defensins, S100 proteins) in dermal keratinocytes [26][27][28].The present signaling synergy was so profound that Il22ra1Il17ra -/-mice subjected to OPC required sacrifice, which was almost never seen in individual knockouts [17,23].Although the basis for their mortality is uncertain, likely contributing factors are severe oropharyngeal/esophageal inflammation that impairs nutritional intake (evidenced by weight loss), and potentially fatal dissemination of intestinal bacteria, described recently by Drummond and colleagues [29].
SPRRs are best understood as precursors for the cornified envelope in stratified squamous epithelia [21,30], prominently expressed in conditions of hyperkeratinization and dermal inflammation [31], but are now appreciated to have important antimicrobial activities.For example, mice lacking Sprr1a and Sprr2a show increased susceptibility to MRSA and dermal P. aeruginosa infections [32].SPRR2A causes membrane disruption in several bacterial species, many of which reside in the oral cavity, and also limits bacterial adherence to gastrointestinal epithelium [21,32].A recent report demonstrated anti-helminth properties of SPRRs [25].We show that at least 1 family member (SPRR2A) exhibits direct anti-Candida activities in vitro, and SPRRs contribute to control of candidiasis in vivo.Although expression of SPRRs at early time points (day 2) was strongly IL-17/IL-22-dependent, evidently other inflammatory events associated with high fungal loads drive expression at later times (day 7).To our knowledge, this is the first connection of SPRRs to fungal host defense, and future studies may determine whether direct application of SPRRs to the oral mucosa can be exploited as an antifungal therapy.
Our assessment of SPRR2a (one of the few SPRRs for which detection reagents are available), showed prominent expression in the oral epithelium during OPC.Oral epithelial cells (OECs) are key responders to Type 17 cytokines, yet there is a clear separation of cytokineresponsive cell types within this tissue [17,36,37].Specifically, while IL-17 signals in the superficial, K13 + post-mitotic OEC layer via the IκBz transcription factor [23,36], IL-22 is required mainly on the proliferating K14 + basal stem-like cell layer [17,36].Admittedly, this view of oral epithelial dynamics is overly simplistic, as cells in the basal layer undergo complex transitional states during differentiation [38].More in-depth characterization of how and where antifungal effectors such as SPRRs are regulated during candidiasis is warranted.
In summary, SPRRs, in part regulated by Type 17 pathway cytokines, make clear contributions to oral antifungal defense.Whether SPRRs play roles in other forms of candidiasis remains to be determined.These observations may have clinical utility, given the emergence of multidrug-resistant fungal strains and the unmet need for strategies to target such infections [1,5].conducted using default parameters.Data are available at the Sequence Read Archive http:// www.ncbi.nlm.nih.gov/bioproject/1094974.